Reaction of amines with functionalized polymers
Covalent coupling of amines, which may be bioactive compounds, e.g. proteins, onto functionalized polymeric surfaces, is commonly accomplished by interacting amine groups of the proteins with desired functional groups of the polymers.
In particular, the binding of amino ligands, e.g. proteins, to polymers containing aldehyde groups (polyaldehyde polymers) is depicted in FIG. 1. The Schiff base products are unstable in aqueous solution since they are in equilibrium with the interacting reagents, but they may be stabilized, e.g. by reduction of the Schiff base bonds with an appropriate reducing agent, e.g. NaBH4.
Dialdehyde dextran and dialdehyde cellulose are examples of polyaldehyde polymers commonly used for immobilization of proteins through the formation of Schiff base products. These polymers are commonly obtained by periodate oxidation of vicinal hydroxyl groups of the parent polymers, dextran and cellulose, respectively, see J. Turkova, et al, Can. Pat. 1217134 (1987). The aldehyde content of these polymers can be controlled by oxidation conditions, i.e. periodate concentration. The backbone of these dialdehyde polymers has a completely different structure than that of the parent polymers (FIG. 2 ). This difference results in a more open structure, higher porosity, increased water solubility, increased biodegradability and decreased mechanical strength of the dialdehyde polymers relative to its parent polymers. For example, cellulose is considered to be a non-biodegradable polymer while dialdehyde cellulose is a biodegradable polymer, a property which is desired for controlled release studies (see M. Singh, Vasudevan, T. J. M. Sinha, A. R. Ray, M. M. Misro and K. Guha, J. of Biomed. Mat. Res. 15, 655 (1981)), but undesired for other biomedical applications, e.g. wounds treatment.
The Schiff base bonds formed by the interaction of dialdehyde polymers with proteins are not stable in aqueous solution (FIGS. 1 and 2), resulting in a leakage of the bound protein. Furthermore, the reaction of proteins with polyaldehyde polymers is usually incomplete, approximately 0.5%-10% of free aldehyde groups participating, see S. Margel and E. Wiesel, J. Polym. Sci., Chem. Ed. 22, 145 (1984). Therefore, residual aldehyde may intramolecularly interact with amino groups of the bound protein, thus significantly decreasing its activity, see S. Margel, J. of Chromatography, 462, 177 (1989). Blocking of residual aldehyde groups and stabilization of Schiff base bonds may be effected by reduction with reducing agents, e.g. NaBH4 (FIGS. 1 and 2). However, the reduced polymers possess significantly decreased mechanical strength, higher water solubility and frequently significantly decreased protein activity, due to reduction of bonds such as disulfide required for native protein activity, see L. Peng, G. J. Calton and J. B. Burnett, Applied Biochem. and Biotechn. 14, 91 (1987).
FIG. 3 describes the binding chemistry of proteins to polymers containing free hydroxy groups, e.g. cellulose, agarose, dextran, etc. This binding method is based on the activation of hydroxyl groups of the polymeric matrix by reaction with various reagents, e.g. cyanogen bromide, tosyl chloride, tresyl chloride, etc., see E. V. Groman, and M. Wilchek, TIBTECH, 5, 220 (1987). The activated polymer is then used for covalent binding of amino ligands (and thiol ligands), e.g. proteins, by nucleophilc-substitution reaction, according to FIG. 3.
The binding of amino (or thiol) ligands to polymers containing hydroxyl groups according to such a derivatization method has been intensively investigated, see K. Nilsson, K. Mosbach, Eur. J. Biochem., 112, 397 (1980). Generally, this reaction is accomplished by adding the dried polymer to an organic solvent (e.g. acetone or dioxane) containing a desired concentration of activating reagent (e.g. tosyl chloride, tresyl chloride, etc). A base (e.g. pyridine, triethylamine, etc) is then added to the organic solution in order to neutralize liberated HCl. The polymer is then washed with an appropriate organic solvent from an unbound activating reagent. The dried activated polymer is then reacted in aqueous solution with the desired amino (or thiol) ligand. Any unbound ligand may then be removed. If necessary, residual tosylate groups may be blocked by known methods, e.g. basic conditions or a reaction with a second amino (or thiol) ligand. The common organic bases used fop neutralizing liberated HCl in this activation procedure are pyridine and triethylamine. A. R. Comfort, E. C. Albert and R. Langer, Biotech. and Bioeng. 34, 1366 (1989), demonstrated that the retention activity of heparinase bound to cellulose by tresyl chloride activation increased by threefold if triethylamine was used as organic base instead of pyridine.
The reaction of primary hydroxyl groups with p-toluene sulfonyl chloride (tosyl chloride) and/or trifluoroethane sulfonyl chloride (tresyl chloride) forms tosylate esters, which have excellent reactivity with amino (and thiol) ligands, as illustrated in FIG. 4. The structure of the cellulose backbone does not change during this immobilization method (FIG. 5), thereby its basic chemical and physical properties, i.e. solubility, mechanical properties, non-biodegradability, etc, remain almost unchanged. Furthermore, the chemical bonds formed by the above activation reagents, e.g. tosyl chloride and tresyl chloride, are stable, thus preventing any significant leakage of bound protein into the solvent, see S. P. Colowick and N. O. Kaplan in "Methods in Enzymology" 135-B, 29 (1987).